This invention relates to novel compounds derived from amylin. The invention also relates to pharmaceutical compositions containing such compounds and their use in the treatment of bone disorders where stimulation of bone growth is required.
Amylin is a 37-amino acid peptide co-secreted with insulin from the beta cells of the pancreatic islets. It is structurally related to calcitonin gene-related peptide (CGRP) and more distantly to calcitonin itself. As a result of these similarities and the fact that calcitonin has potent effects on osteoclasts, the actions of amylin in bone have been studied. Amylin mimics some of the effects of calcitonin on bone resorption. It stimulates cyclic AMP formation and induces quiescence in osteoclasts, reducing the amount of bone resorbed. Similar effects are demonstrable in bone culture.
Amylin was first reported by Cooper et al in Proceedings of the National Academy of Sciences USA 84, 8628 (1987) and is the subject of European Patent 289287.
The native molecule contains a disulphide bridge between the cysteine residues shown at positions 2 and 7 in the primary structure. Further, it is amidated at the 3xe2x80x2-end and it is formed as a propeptide.
European Patent 289287 reports a number of novel biological effects including enhancement of hepatic glucose output, increased production of lactate from skeletal muscle and reduced action of insulin in skeletal muscle.
Amylin is also reported in European Patent 408284 as having value for treatment of bone disorders and calcium imbalance. The patent specification attributes the activity of amylin to an inhibition of osteoclast motility.
The use of amylin and amylin agonists for the stimulation of bone growth is described and claimed in our related patent application PCT NZ 95/00062 (published as WO 96/62269).
Amylin has been found to affect osteoblasts. There is a dose-dependent stimulation of osteoblast proliferation by amylin in concentrations as low as 10xe2x88x9211M. Histomorphometric indices of bone formation are increased in vivo following local injection of the peptide. The anti-osteoclastic potency of amylin is substantially reduced in the absence of the carboxyl terminal amide group.
However, details of the structure-activity relationships for the bone effects of amylin are unclear. The actions of amylin to stimulate bone formation and inhibit bone resorption make it an attractive candidate for the therapy of osteoporosis and for the promotion of the repair of local bone defects. The full peptide would be expensive to manufacture, not be active orally, and might have deleterious effects on carbohydrate metabolism.
Amylin has been implicated in the pathogenesis of type 2 diabetes, in part, as a result of amyloid deposition in the pancreatic islets. However, the action of amylin on carbohydrate metabolism requires the intact peptide. Its propensity to form amyloid is dependent, in part, on the amino acids between positions 25 and 29. Thus a peptide that exhibits osteoblast proliferative effects and which has no effect on carbohydrate metabolism or a tendency to lead to amyloid formation is highly desirable as a stimulant of bone formation in the management of osteoporosis and other bone disorders.
While intact amylin produces the greatest maximal effect on osteoblast proliferation, we have now surprisingly found that N-terminal amylin-(1-8), also stimulates osteoblast proliferation, without the effects on carbohydrate metabolism or a tendency to lead to amyloid formation possessed by the whole peptide.
The invention provides compounds which are useful for stimulating bone growth in an animal and is useful for preventing bone deterioration in an animal.
The invention also provides a pharmaceutical composition containing such compounds together with a pharmaceutically acceptable carrier preferably in orally administrable form.
The compounds of this invention are those of formula I.
R1-Cys-Asn-Thr-Ala-Thr-Cys-R2 (SEQ ID NO:2)xe2x80x83xe2x80x83(I)
where R1 is absent, Lys or a functionally effective group and R2 is absent, or Ala or a functionally effective group; or a functionally effective derivative thereof.
The invention also provides a method of stimulating bone growth comprising subjecting bone to the effect of an effective amount of a compound of formula I. 
The method of the invention will be particularly useful for treatment of osteoporosis of any cause, bone loss or malignancy, or endocrine disorders or immobility and disuse and in fractures by enabling the patient to reverse previous bone loss. The method can be used for treatment of bone disorders in all animals e.g., mammals particularly humans, cattle, horses, dogs and cats.
The invention further provides a method of prophylactically increasing or maintaining bone density in a subject having a substantially normal bone density comprising the step of administering an effective amount of the amylin fragment.
By this aspect of the invention it is envisaged that patients at risk of bone deterioration will be given a regular dose of the amylin fragment to prevent likely bone deterioration. Patients at most risk are post menopausal women, usually at age above 50, and men over 60 years of age.
Administration within the scope of the invention can also be after bone density has deteriorated beyond the normal level. Normal bone density means bone density within two standard deviations of the mean value for race, age and sex. In view of the effect of inhibiting osteoclast activity and the effect of stimulating bone growth, treatment with the amylin fragment is capable of reversing bone loss.
The compounds of this invention are the 2 to 7 amino acids of amylin with a disulphide bond between the 2 and 7 cysteine groups. Amylin (1-8) is active while amylin (1-8) with 2 to 7 disulphide bond removed is inactive and blocks the activity of amylin (1-8). The 2 to 7 disulphide bond is therefore necessary for osteoblast proliferation.
The N-terminal group R1 can be absent but is preferably lyseine. Functionally effective groups as R1 include derivatives of the N-terminus eg acid addition salts and one or more alternative amino acids and derivatives thereof in which the compound has osteoblast stimulating activity. Similarly the C-terminal group R2 can be absent but is preferably alanine and is preferably amidated. Functionally effective groups as R2 include derivatives such as amides and one or more alternative amino acids and derivative thereof where the compound has osteoblast stimulating activity without an effect on carbohydrate metabolism or a tendency to lead to amyloid formation.
Functionally effective derivatives of the compounds of formula I include agonists and related peptides in which one or more of the 2 to 7 amino acids are replaced by other amino acids and which stimulate osteoblast activity without an effect on carbohydrate metabolism or a tendency to lead to amyloid formation.
The compounds of the invention can be produced by known methods but are preferably synthesised using standard solid phase peptide synthesis techniques. For example, the compounds can be prepared on methylbenzyhydrylamine resin followed by hydrogen fluoride deprotection and cleavage from the resin.
For use in therapy, the compounds can be utilised by themselves or as functionally effective derivatives. The compounds have amine groups which can form salts in accordance with the invention. All the compounds have at least one amino group which will be capable of forming a salt. Where two or more amino groups are present in the compound, the possibility of mixed salts exists which are also within the scope of the invention.
The compounds can be used in the form of a salt with an acid. Acids which can be used preferably include compatible inorganic acids such as hydrochloric and organic acids (or salts thereof) more preferably those occurring in living organisms, including but not limited to oxalic acid, glucuronic acid, pyruvic acid, lactic acid, citric acid, isocitric acid, ketoglutaric acid, succinic acid, malic acid, and oxaloacetic acid. In the preferred case of an aqueous solution, the desired anion can be added either as the free acid, or a salt, preferably one which is highly soluble in water, for example the sodium or potassium salts, but also the lithium, magnesium, calcium or ammonium salts. Moreover, these salts can be used either in anhydrous or hydrated forms. For example citric acid can be used as the anhydrous free acid, the monohydrate free acid, the anhydrous trisodium salt, or the dihydrate trisodium salt. These salts can be prepared by the methods described in WO 96/02269. The compounds can also be amidated at the C-terminal end.
It is desirable that all trifluoroacetic acid or other bone inhibitory acetic acid anions be removed or alternatively be present in such minor amounts as to have no therapeutic effect.
The compounds of the invention can be formulated into pharmaceutical compositions in the normal way to make oral, intranasal or parenteral formulations dependent upon the desired form of administration of the substance. Preferably the compounds can be formulated in orally administrable form eg as tablets, capsules suspensions, syrups and the like.
The compound is administered in an amount to meet the particular condition under treatment. It will be administered to the mammal either injectably, intranasally or in an oral formulation in a form for ensuring the availability of the compound at the particular site for effective therapy. The compounds of this invention are expected to be effective orally.
Parenteral administration can also be employed, either subcutaneously or intramuscularly. For the treatment of fractures or other localised bone disorders, application of the compound directly to the site may be desirable, for example, by injection or by application during surgery. It is believed that the dosage administered will lie within the range 0.01-100 mg/kg of body weight. The actual dose administered to each patient will depend on the type of patient and the nature of the disorder being treated. It is envisaged that for prophylactic therapy the dose will be lower than that required for reversal of bone resorption or treatment of fractures.
Administration of two or more compounds according to the invention, is within the scope of the invention as is the use of a compound of the invention with any other effective therapeutic agent (e.g., calcitonin) including any other agent for treatment of bone disorders. Combination agent therapy can be by separate administration of the individual agents or by combining the two or more agents into one composition form.